Abstract: Oxidative stress caused by elevated intracellular reactive oxygen species (ROS) has been linked to various conditions, including Parkinson's diseases, amyotrophic lateral sclerosis, and cancer. Non-invasive approaches to quantify tissue ROS may allow for early diagnosis, disease stratification, and evaluation of treatment responses. Given that highly reactive ROS has an extremely short lifetime (in s) and low in vivo concentrations (M level), it has proven difficult to measure ROS directly. Current approaches for detecting ROS or tissue redox activity include biochemical analyses, optical redox scanning, electron paramagnetic resonance (EPR) imaging, and contrast-based MRI; however, each of these approaches is either invasive or requires the use of exogenous contrast agents. To date, there is no non-invasive imaging approach to assess in vivo ROS production based on endogenous contrast. Using an imaging phantom that continuously produces hydroxyl ROS (fresh egg-white tissue treated with H2O2), we discovered several novel endogenous MRI properties of ROS, including promotion of proton exchange, reduction of CEST contrast, and shortening tissue T1-relaxation time. Based on our preliminary findings, endogenous ROS MRI appears to be a promising and novel method of taking pictures of short-lived ROS in living tissues. Our central hypothesis is that endogenous ROS MRI has the sensitivity and specificity to detect ROS changes in vivo in response to various oxidative challenges and antioxidative therapies. To test this hypothesis, two specific aims are proposed. To test this hypothesis, three specific aims are proposed. Aim 1: Determine the sensitivity and specificity of endogenous ROS MRI. Detection sensitivity of in vivo ROS production will be characterized with an H2O2-treated wound animal model. Confounding factors such as changes in metabolites and metabolite concentrations will be further investigated and ruled out. In addition, magnetic field dependency of ROS MRI and other species of free radicals will be studied. Aim 2: Image ROS changes in vivo in response to oxidative challenges and antioxidant treatment using endogenous ROS MRI. In this aim, we will validate ROS MRI preclinically in rats exposed to rotenone challenges. We will also evaluate the response to the antioxidant lipoic acid. In total, 12 groups of rats will be treated with vehicle, rotenone, or lipoic acid and then undergo behavioral analysis, ROS MRI, and ex vivo histopathological studies. Aim 3: Determine pro- oxidant cancer drug selectivity with endogenous ROS MRI. Endogenous ROS MRI will be used to longitudinally image the responses of pro-oxidant chemotherapies in both tumor and brain, providing information about the selectivity of cancer chemotherapies. 6 groups of F98 glioma bearing rats treated with drugs with high or low selectivity (Piperlongumine and rotenone) will be studied. The successful completion of this study will establish a novel MRI technique as a longitudinal imaging tool for non-invasively quantifying and mapping ROS production in normal and disease conditions with high sensitivity and specificity.